Thioarsenate Toxicity and Tolerance in the Model System

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Thioarsenate toxicity and tolerance in the model system Arabidopsis thaliana Britta Planer-Friedrich, Tanja Kühnlenz, Dipti Halder, Regina Lohmayer, Nathaniel Wilson, Colleen Rafferty, and Stephan Clemens Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 19 May 2017 Downloaded from http://pubs.acs.org on May 21, 2017

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Environmental Science & Technology

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Thioarsenate toxicity and tolerance in the model system Arabidopsis thaliana

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Britta Planer-Friedrich1*, Tanja Kühnlenz2, Dipti Halder1, Regina Lohmayer1, Nathaniel Wilson3, Colleen Rafferty2, Stephan Clemens2 1

Environmental Geochemistry, Bayreuth Center for Ecology and Environmental Research (BayCEER),

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University Bayreuth, Universitätsstraße 30, 95447 Bayreuth, Germany

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2

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Germany

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Department of Plant Physiology, University of Bayreuth, Universitätsstraße 30, 95447 Bayreuth,

formerly at Environmental Geochemistry, Bayreuth Center for Ecology and Environmental Research

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(BayCEER), University Bayreuth, Universitätsstraße 30, 95447 Bayreuth, Germany; now at Babbage

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Consultants Limited, 1010 Auckland, New Zealand

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* corresponding author: address: Environmental Geochemistry, Bayreuth Center for Ecology and

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Environmental Research (BayCEER), University Bayreuth, Universitätsstraße 30, 95447 Bayreuth,

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Germany; phone: +49 921 553999, fax: +49 921 552334; email: [email protected]

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1 Environment ACS Paragon Plus


Col-0 As

As

=

cad1-3 As

PC > As

PC
11, so it is generally not environmentally relevant 48.

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Investigating thioarsenates is difficult because the species are pH- and oxygen-sensitive and require

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specific stabilization (flash-freezing 33 or, in the presence of iron, anoxic storage with ethanol addition

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Acidification induces AsS-mineral precipitations

or SPE-based separation 50) and analytical techniques (chromatographic separation at pH 12-13 33). 51

and the transformation of thioarsenates to

48

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arsenite or arsenate . Therefore, most studies to date only analyze for arsenite and arsenate even

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in the presence of free sulfide, e.g. Jia and Bao

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incubations amended with 100 mM sulfate at pH 7.2 but did not look for thioarsenates. Interestingly,

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in his study on microbially catalyzed As transformation in the rice rhizosphere, Somenahally

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reported an unidentified As species eluting after arsenate which he speculated was

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monothioarsenate. In preliminary studies, we have, for the first time, been able to detect mono- and

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dithioarsenate in paddy soils, both under natural conditions in the field (maximum 10% of total As),

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as well as in laboratory incubation experiments mimicking sulfate fertilization (maximum 63% of total

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As).

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who detected 1.4 mM sulfide in paddy soil


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In summary, both geochemical considerations and direct analytical evidence suggest that

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thioarsenates form in rice paddy soils, and previous studies confirmed that they can even occur in

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the presence of iron. Moreover, thioarsenates are expected to show less sorption than arsenite and

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arsenate to iron plaque in the rhizosphere and so they may be more bioavailable for uptake into the

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rice plant.

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There is, to date, no information about uptake, accumulation, toxicity or tolerance of any

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thioarsenate species in plants. We therefore aimed to obtain the first mechanistic insights into the

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potential toxicity of monothioarsenate (in comparison to its structural oxyanion analogue arsenate),

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as the most stable and – based on our own preliminary studies – the dominant thioarsenate species

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in soil incubations under sulfate-reducing conditions. We chose the model plant A. thaliana for this

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pilot study because of ease of cultivation and, more importantly, the availability of very useful

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biological material such as defined mutants for relevant pathways. Many components of As transport

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and metabolism in plants have been identified and characterized in A. thaliana. For instance, besides

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the wild type (Col-0), a GSH-deficient (cad2) mutant, a PC-deficient mutant (cad1-3), and a double

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knock-out mutant for the PC vacuolar transporters (abcc12) are available 53. Direct comparison of

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Col-0 and these mutants enables direct investigations on the potential role of PCs in detoxification of

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thioarsenic species. For rice, such mutants are not readily available, yet. The toxicity was tested by

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growing A. thaliana on agar plates for tolerance assays and in hydroponic cultures containing

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arsenate and monothioarsenate, measuring growth parameters such as root length and seedling

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fresh weight as well as accumulation of As and formation of PCs in roots and shoots.

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MATERIALS AND METHODS

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Plant growth and As treatment. The A. thaliana wild-type Col-0 and the AtPCS1 mutant cad1-3

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were used for testing toxicity of monothioarsenate on plant growth. Before use in As tolerance

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assays on agar plates, or cultivation in hydroponic culture, A. thaliana seeds were surface-sterilized

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by exposure to chlorine gas for 35 min. The medium used for plant growth was based on the

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modified Hoagland's solution No 2 as described previously 55 (0.28 mM Ca(NO3)2, 0.6 mM KNO3, 0.1

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mM NH4H2PO4, 0.2 mM MgSO4, 4.63 µM H3BO3, 32 nM CuSO4, 915 nM MnCl2, 77 nM ZnSO4, 11 nM

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MoO3). According to Chaney 56, Fe was supplied as N,N′-di-(2-hydroxybenzoyl)-ethylenediamine-N,N′-

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diacetic acid (Fe-HBED) to a final concentration of 5 µM.

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For tolerance assays on vertical agar plates, medium containing 1% (w/v) sucrose and 0.05% (w/v) 2-

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(N-morpholino)ethanesulfonic acid (MES) at pH 5.7 that was devoid of microelements, except for Fe,

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was used. Arsenate [sodium arsenate dibasic heptahydrate (Na2HAsO4 × 7H2O, Fluka)] and

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monothioarsenate [(Na3AsVO3S × 7 H2O, synthesized in our laboratory according to a previously


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described method

; purity 97%, the remainder being arsenate] were added at indicated

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concentrations between 1 and 200 µM. For comparison, 5 µM arsenite (AsCl3, Sigma-Aldrich, St Louis,

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MO, USA) were also tested. The medium was solidified with 1% (w/v) Agar Type A (Sigma-Aldrich, St

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Louis, MO, USA). Plates were sealed with Leucopore tape (Duchefa Biochemie, Haarlem, NLD)

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followed by stratification for 2 d at 4°C. For plant growth, plates were then incubated for 14 d under

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long-day conditions (16 h light/ 8 h darkness) at 23°C and 75 µE light intensity. After 14 days, primary

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root length and seedling fresh weight was determined. To investigate the PC-based detoxification

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pathway in greater detail, tolerance assays were not only conducted with Col-0 and cad1-3 but also

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with the mutants cad2 (a GSH biosynthesis mutant)

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mutant) 25.

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For analysis of PC and As contents, plants were grown hydroponically for 6 weeks in medium without

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sucrose, containing 0.05% (w/v) MES and microelements at pH 5.7 as described before

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stratification, cultivation started in agar-filled PCR tubes in pipette tip boxes for 3 weeks, followed by

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a transfer into 50 mL tubes (Greiner Bio-One, Kremsmuenster, AUT) for another 3 weeks under short-

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day conditions (8 h light at 22°C/ 16 h darkness at 17°C) at a light intensity of 150 µE. Medium was

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changed weekly in order to guarantee a sufficient mineral and oxygen supply. Addition of As species

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started after 5.5 weeks at approximately same-effect concentrations of 200 µM to Col-0 and 20 μM

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to cad1-3. Arsenic-exposure lasted for 4 days. The stability of arsenate and monothioarsenate in the

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externally applied medium was confirmed by analyzing test solutions (without plants) at day 0 and

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day 4 (Table S1) by ion chromatography (Dionex ICS-3000; AG/AS16 IonPac column, 20-100 mM

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NaOH at a flow rate of 1.2 mL/min) coupled to the ICP-MS as described earlier 33. Due to a lack of a

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commercial standard, monothioarsenate was quantified via the arsenate (Na2HAsO4×7H2O, Fluka)

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calibration curve. The validity of this approach has been shown in a previous publication 33.

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To investigate the extent of efflux, Col-0 plants that had been grown for 14 days in hydroponic

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culture at 6.7 µM arsenate and monothioarsenate, respectively, and showed no signs of reduced

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plant growth, were transferred to As-free medium. After 0, 1, 2, 4, 7, 9, and 11 days 1 mL of aqueous

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medium was removed and analyzed for total As by inductively coupled plasma-mass spectrometry

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(ICP-MS, XSeries2, Thermo-Fisher) using oxygen as reaction cell gas (AsO+, m/z 91). The percentage of

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As released was calculated by multiplying the measured As concentrations in solution with the

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respective medium volume at each sampling time and referring this absolute concentration to the

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original concentration of total As stored in the plants after the initial 14 days of As-exposure.

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Analysis of total As and PCs in roots and shoots. Roots were first carefully washed with distilled

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water, followed by 2 washing steps with 0.1 M sodium phosphate buffer solution (pH 5.7) and a final

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washing step with water. Each washing step was performed with 25 mL per root sample at 4°C for 10

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and abcc12 (a PC vacuolar transport double


55

. After


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minutes to remove any surface-bound As. Frozen root and shoot samples were ground in liquid

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nitrogen and the powder stored in the freezer until analysis.

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For the determination of total As, approximately 100 mg of sample were digested in 3 mL

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concentrated HNO3 and 2 ml 30% H2O2 in a CEM Mars 5 microwave digestion system (CEM

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Corporation, Matthews, NC, USA). Digested samples were analyzed for total As by ICP-MS as

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described above. The microwave blank (HNO3/H2O2) was subtracted from all concentrations and

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results are reported as mean concentrations with standard deviation of three biological replicates.

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PCs were determined from pooled root samples and pooled leaves samples including shoots by

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UPLC-ESI-QTOF-MS as described earlier 55. Briefly, plant material was frozen in liquid nitrogen and

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ground to homogenous powder followed by extraction with 0.1 % (v/v) trifluoroacetic acid (TFA)

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containing 6.3 mM diethylene triamine pentaacetic acid (DTPA) and 40.04 µM N-acetylcysteine (NAC)

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as internal standard. Before derivatization with monobromobimane (mBrb) at 45°C for 30 min, thiol

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groups were reduced with Tris-(2-carboxyethyl)-phosphine (TCEP). The separation of the mBrb-

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labelled thiols was performed by a Waters Aquity UPLC systems equipped with a HSS T3 column (1.8

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μm, 2.1 × 100 mm; Waters Corporation, Milford, MA, USA) at an injection volume of 5 µL and the

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linear binary gradient of water (A) and acetonitrile (B), both acidified with 0.1% (v/v) formic acid, at a

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flow of 0.5 mL min–1: 99.5 % A, 0.5 % B for 1 min, a linear gradient to 60.5 % B at 10 min, gradient to

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99.5 % B at 12 min, flushing with 99.5 % B for 1 min, a gradient back to initial conditions in 1 min and

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an additional re-equilibration for 1 min. The column temperature was set to 40°C. Thiols were

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detected with a Q-TOF Premier mass spectrometer equipped with an ESI-source (Waters

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Corporation) operated in the V+ mode. Data were acquired from m/z 300–2000 with a scan time of

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0.3 s and an inter-scan delay of 0.05 s. For quantification the QuanLynx module of the MarkerLynx

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software was used. All samples were measured in three technical replicates.

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Statistical analysis. Root lengths and seedling fresh weights data were statistically analyzed by pair-

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wise analysis using post-hoc analysis (Tukey Honest Significant Differences (TukeyHSD)) following

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two-way ANOVA (significance defined as p 1 µM upon exposure to monothioarsenate and

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> 5 µM upon exposure to arsenate (Figure 1b, d and Figure S2b, d). At concentrations above 100 µM,

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where Col-0 still had less than 50% growth reduction, cad1-3 was not able to germinate anymore.

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The IC50 value calculated from the dose response curve of relative root length versus individual As

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species concentration (Figure S4) was 14.9 ± 0.8 µM and 16.7 ± 1.2 µM for cad1-3 for exposure to

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monothioarsenate and to arsenate, respectively. The result of an almost 10 times lower tolerance of

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cad1-3 compared to Col-0 observed in our study is, at least for arsenate, comparable to previous

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results by Ha et al. 53 and Liu et al. 23 who assessed the role of PC formation in arsenite and arsenate

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tolerance. For monothioarsenate, our results are the first direct evidence that PC formation plays an

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important role in thioarsenate detoxification as well.

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Along with significantly lower growth rates, the arsenic concentration in the roots was 15 times

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lower in cad 1-3 than in Col-0 for exposure to arsenate, and below detection limit (factor > 50) in cad

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1-3 exposed to monothioarsenate. The higher accumulation of As in the roots of Col-0 gives further

12, 13


, enzymatic monothioarsenate

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and also in the present


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indirect evidence for efficient As-PC complexation and vacuolar sequestration in the roots as it has

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been described before 23.

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Direct analytical evidence for PC formation was found in the roots (Figure 4a) and, to a lesser extent,

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also in the shoots (Figure 4b) of Col-0 both upon exposure to arsenate and monothioarsenate.

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Analysis using MANOVA indicates that there is a significant relationship between arsenic species and

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plant genotype with both PC2 and PC3 formation. Absolute PC concentrations were significantly

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lower after exposure to monothioarsenate compared to exposure to arsenate both in the roots and

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in the shoots (p0.05), while

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concentration of PC3 was significantly higher (350 mg/kg fw) than PC2 (172 mg/kg fw) for exposure

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to monothioarsenate (p

Thioarsenate Toxicity and Tolerance in the Model System

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